Spatial transcriptomics reveals molecular mechanisms underlying neuroinflammatory and neurodegenerative sequelae of immune checkpoint inhibition [RNA-Seq]

Immune checkpoint inhibitors (ICIs) that block cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) signaling are among the most promising therapeutic approaches in tumor immunotherapy. We demonstrated that ICI disrupts synaptic integrity and neuronal plasticity by adversely altering microglial and neuro-immune homeostasis. However, the cell type–specific molecular mechanisms underlying ICI-induced neuroinflammation and neuronal dysfunction remain poorly defined. To address this gap, we performed spatial transcriptomic profiling in a syngeneic melanoma mouse model using multiplexed error-robust fluorescence in situ hybridization (MERFISH) to map gene expression at single-cell resolution. By integrating spatial single-cell data with bulk RNA-seq from the hippocampus, we characterized the distribution of microglia, astrocytes, synaptic, and neuroinflammatory markers and determined how ICI reshapes hippocampal cellular composition in cancer-bearing and non-cancer mice brains. MERFISH revealed upregulation of microglial, astrocytic, oligodendrocytic and T cell markers post-ICI treatment, implicating pathways involved in neuroinflammation, synaptic function, and cellular signaling. Immunofluorescence analysis corroborated these findings in postmortem brains from patients treated with ICIs and demonstrated elevated microglial activation and astrogliosis, along with reduced expression of myelin sheath stability markers. Mechanistically, conditional T cell depletion in a transgenic mouse model revealed that ICI-mediated microglial activation is contingent on the presence of T cells, thus establishing a causal T cell-microglia crosstalk axis driving neuroinflammation. Collectively, this work provides a high-resolution framework for understanding ICI treatment-associated neurotoxicity and establishing a significant link with neuroinflammation-associated cognitive decline, while also revealing potential molecular targets for therapeutic intervention.